The present invention generally relates to the determination of log P values for libraries of compounds, for example, combinatorial and/or lead generation libraries.
For many drugs and pesticides, there is a relationship between their physiological activity and their hydrophobicity. The classical method for determining the hydrophobicity of a compound is to determine the logarithm of the partition of the compound in water and n-octanol. When a relationship between chromatographic retention data and logarithm of n-octanol/water partition coefficients (Log P) can be established, the hydrophobicity of a compound can be determined chromatographically rather than using the shake flask method. The n-octanol/water partition system is not necessarily an ideal one. Chromatographically derived measures can be even more reliable, depending on the reliability of the particular HPLC method.
There are advantages to using a chromatographic method versus the shake flask method. For example, the chromatographic method is faster and more suitable for compounds containing impurities. No quantitative measure is required, and the method can be applied to volatile compounds.
Numerous HPLC methods for determining Log P values have been developed. One of the limitations associated with using HPLC is that special care must be taken when one is evaluating ionizable compounds. Often, the calculation of the Log P requires a correction for the dissociation factor (CD) for the compound.
One method for determining Log P using an HPLC method involves coating a silanized Kieselguhr support with water-saturated n-octanol and using an n-octanol saturated buffer as the eluent. Mirrlees et al., J. Med. Chem., 19:615 (1976). Log P values in the range of between xe2x88x920.3 and 3.7 can be obtained by varying the column length and flow rate. Correlations have also been made between Log P and the log of the retention volume on a C18 column. Numerous other means for correlating Log P and HPLC are known, each of which has certain advantages and disadvantages. Chemical Analysis, 91:234-278 (Chapter 11), Wiley and Sons, New York, (1987)
Retention parameters obtained from HPLC measurements (log kxe2x80x2) can be used to obtain information regarding the hydrophobicity of compounds. The retention data obtained with a hydrophobic stationary phase and using water as the mobile phase can be used directly, although this typically results in extended retention times. Isocratic data, measured at certain eluent compositions (typically as log kxe2x80x2x, for a given eluent composition X), can be used to calculate Log P data. Also, when using aqueous solutions which contain water miscible organic solvents such as methanol, acetonitrile, and acetone, chromatographic data can be extrapolated to the point at which there is 0% organic solvent in the mobile phase, which permits one to do the HPLC runs faster than when water alone is used and still obtain reasonably reliable results.
However, the method tends to be limited in that only a limited amount of water-miscible organic solvents can be present in the water. If too much organic solvent is present, the linear relationship between the concentration and the log kxe2x80x2w may no longer exist. Several authors have advocated using isocratic log kxe2x80x2x values rather than log kxe2x80x2w values.
A further limitation of classical HPLC methods for determining Log P values is that they often rely on correlation between the capacity factor and Log P, and this correlation is typically close only for related compounds, and is poorer for compounds containing different functional groups.
In spite of the limitations associated with the use of too much organic solvent and the problems associated with analyzing highly polar ionizable compounds on a reverse phase column, HPLC methods are still applicable for determining Log P values for a large number of organic compounds, based on the linear relationship between log P and log kxe2x80x2. Nevertheless, it would be advantageous to provide additional HPLC methods which overcome the limitations of these methods. Further, it would be advantageous to provide useful methods for determining the hydrophobicity of libraries of compounds. The present invention provides such methods.
Methods and apparatus for high through Log P determination of libraries of compounds, for example, combinatorial or lead generation libraries, are disclosed. The methods involve obtaining a number of compounds to be evaluated, and using an HPLC method to determine the Log P value for the compounds. In a preferred embodiment, the purity and Log P values for the compounds are determined simultaneously. In another preferred embodiment, the compounds are subjected to Log P determination and bioassay for determination of their bioactivity. Compounds with desired bioactivity and Log P can be subject to further examination, and evaluated separately from compounds which have the desired Log P but not the desired activity and compounds which have the desired activity but not the desired Log P.
The method is advantageous in that it allows rapid determination of compounds not only with the desired bioactivity but also with the desired hydrophobicity/hydrophilicity for the intended use. Large numbers of compounds in combinatorial libraries can be screened and the most effective compounds for a particular indication rapidly identified.
In another preferred embodiment, the method allows the Log P determination to be calculated based on retention time, rather than an isocratic method (which does not use a gradient).